In vitro culturing techniques have been used to evaluate the chemistry and physiology of avian development and morphogenesis. Initial techniques were developed for the culturing of 3 day fowl embryos in plastic containers, Dunn and Boon, Poultry Sci. 55: 1067-1071 (1976) and Dunn and Boone, Poultry Sci. 57: 370-377 (1977). The method permits extensive embryonic development but the culture process does not produce viable hatchlings because the embryos show retarded growth and become progressively hypocalcemic. The results of in vitro embryo culture have been improved by culturing embryos in contact with eggshells from which they are able to resorb calcium, Ono and Wakasugi, Poultry Sci. 63: 159-166 (1984). The success of the above techniques has been improved by rocking the surrogate eggshells to produce normal hatchlings, Rowlett and Simkiss, British Poultry Sci. 28:91-101 (1987).
Genetic manipulation of avian primordial germ cells requires a culturing system which will allow the culturing of embryos much smaller than those three days of age. Fertilization (gamete interaction) generally takes place within 15 min of ovulation, with the first cleavage division taking place about 4 hr later, Perry, J. Anat. 150: 99-109 (1987). The ovum begins passage through the oviduct and is invested with albumen in the magnum and with the shell membrane in the isthmus where cleavage commences. Following the first cleavage the cells continue to divide giving rise to a simple sheet of cells overlying a subblastodermal cavity (Kochav et al., Dev. Biol. 79:296-308 (1980). As the soft-shelled egg moves from the oviduct to the uterus the albumen is doubled and the shell undergoes slow calcification. Embryonic morphogenesis and growth take place during the next 21 days and result in fully developed and viable bird that is capable of hatching.
The initial attempt at culturing embryos younger than 3 days was by Perry, Nature 331: 70-71 (1988) and Perry, European Patent Publication No. 295,964, published Dec. 21, (1988). The Perry process requires that the artificially inseminated hens be sacrificed at 2.75 hr after the preceding egg was laid to obtain the fertilized ova. The ova are placed in glass jars and a culture medium was added (culture system I). The culture medium consisted of liquid albumen collected form freshly laid eggs (3 parts) and a salt solution (2 parts) containing (50 mmole KHCO.sub.3, 30 mmole NaHCO.sub.3, 10 mmole KCI, 2.5 mmole MgCl.sub.2.6H.sub.2 O, 0.7 mmole CaCl.sub.2.2H.sub.2 O and 11 mmole glucose. The pH was lowered from an initial value of 8.4 to 7.2-7.4 by stirring in an atmosphere of CO.sub.2. The jars were sealed with Saran Wrap incubated at 41.degree.-42.degree. C. for 24 h. Following incubation the contents of the jars were transferred to recipient egg shells and the shells filled with medium (liquid albumen, 2 parts; salt solution, 1 part), pH 8.2-8.4 and sealed with cling film (culture system II). The eggs of culture system II were rocked intermittently through an angle of 90.degree. in hourly cycles at 38.degree. C., relative humidity (RH) 40-50% for 3 to 6 days. The contents of culture system II are transferred to larger eggshells and the shells are sealed (culture system III). The cultures were incubated at 38.degree. C. , RH 40-60% and rocked intermittently through an angle of 30.degree. in hourly cycles for 5 days, then maintained in a stationary position for the remaining period. The temperature was lowered by 2.degree. C. for the final 3 days. Perforations were made in the cling film cover at 1-2 days before hatching and the cling film was replaced with a lid 12 h before hatching.
Perry's three stage system for the culture of oviducal eggs attempts to mimic the dynamic water and ion fluxes that occur in the shell gland of the fowl during the last 20 h of egg formation. This appears to have been done without an understanding of the critical physiological events that take place during this period of egg formation in fowl. The excessive transfer of the embryos and the limited understanding of the appropriate physiological events resulted in a very low level of successful hatchings by the Perry culture system. This system has been improved by Naito et al., J. Exp. Zool. 254: 322-328 (1990), who completely replaced the thick albumen of the individual eggs with thin albumen and obtained a hatch rate of 34.4%.